scholarly journals Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins

2009 ◽  
Vol 113 (4) ◽  
pp. 855-859 ◽  
Author(s):  
M. Drobizhev ◽  
S. Tillo ◽  
N. S. Makarov ◽  
T. E. Hughes ◽  
A. Rebane

2009 ◽  
Vol 96 (3) ◽  
pp. 400a-401a
Author(s):  
Mikhail Drobizhev ◽  
Shane Tillo ◽  
Nikolay Makarov ◽  
Aleksander Rebane ◽  
Thomas E. Hughes


2022 ◽  
Vol 23 (2) ◽  
pp. 770
Author(s):  
Mikhail Drobizhev ◽  
Rosana S. Molina ◽  
Jacob Franklin

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.



1995 ◽  
Vol 52 (7) ◽  
pp. 4986-4997 ◽  
Author(s):  
Misao Murayama ◽  
Takashi Nakayama


2013 ◽  
Vol 15 (20) ◽  
pp. 7666 ◽  
Author(s):  
Honghua Hu ◽  
Olga V. Przhonska ◽  
Francesca Terenziani ◽  
Anna Painelli ◽  
Dmitry Fishman ◽  
...  


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